Enyimba started this tie without their captain and influential centre-back, Chinedu Udoji, who is recovering from a knock.
In spite of the rainy condition in Owerri, Fidelis Saviour scored what turned out to be the winning goal on 39 minutes.
Heartland toiled for the last 51 minutes to draw level but ended up suffering their second home loss of the season.
Interestingly, Heartland lost their opening game in the first round against Nembe City and have now lost their first match in the second round.
Heartland have now lost in their last five Glo Premier League matches to Enyimba including a 0-1 loss in the reverse game this season.
Enyimba are now fourth in the league standings on 31 points while Heartland have dropped to seventh.
Wikki Tourists were the biggest winners of Matchday 20 when they overran Lobi Stars 4-0.
Ibrahim Shuaibu scored a brace while Ibrahim Babale and Alaba Johnson completed the rout on Dominic Iorfa's men.
Kwara United were close to claiming a third away win of the season in Ibadan against 3SC when Dare Ojo fired in a free kick on the stroke of half time.
Ojo, however, was sent off in the second half and 3SC turned on the screw on Kwara United.
Taofeek Adepoju salvaged a point for 3SC on 67 minutes after Gbolahan Salami's goals in the 52nd and 58th minutes were chalked off for offside.
Bayelsa United needed a late spot kick to earn a point at home after Dolphins goalkeeper, Austin Brown, foiled a number of their goal attempts.
Ifeanyi Egwim put Dolphins ahead in the 12th minute and two minutes later, Samuel Agba put Bayelsa United level.
Dolphins got back in front when Emeka Atuloma rose highest to head away Isiaka Olawale's corner kick.
A spot kick converted by Peter Ebimobowei with 12 minutes left to play ensured the hosts escaped with a 2-2 draw.
A goal in each half from Oghogho Oduokpe and Chinedu Sunday helped troubled Enugu Rangers to a 2-0 win over Nasarawa United. Rangers are now fifth with 30 points and Nasarawa United are 12th.
Sharks gave themselves a boost of escaping the drop by defeating Nembe City 1-0. Tayo Fabiyi got the goal but Sharks remain bottom.
ABS won 1-0 against Kaduna United in Ilorin after Abdulkadir Aminu scored with 15 minutes to the end of the contest. The Ilorin club still remain in the bottom despite the win.
Sunshine Stars and Warri Wolves drew 1-1 in Akure. Oke Ogagatevhwo put the Akure Gunners in front in the 68th minute but ThankGod Ike made sure Wolves got a point with four minutes left to play.
In Uyo, Akwa United raced into a three-goal lead with Mfon Udoh scoring twice and Ubong Ekpai netting once.
Gombe United ensured the game ended on a nervy note for the hosts with goals from Haruna Adamu and Oliver Agbo but Akwa United held on for a 3-2 win.